The cholecystokinin-B/gastrin receptor (CCK-BR) is a seven transmembrane domain peptide hormone receptor. Among its most important physiologic functions, the CCK-BR modulates acid secretion and mucosal proliferation in the stomach as well as anxiety and pain perception in the central nervous system. The potential clinical relevance of this receptor has generated considerable interest in understanding the molecular basis of ligand - CCK-BR interactions. Mutational analysis of the receptor, carried out in our laboratory, suggests he existence of a ligand binding pocket comprised of transmembrane domain residues. Non-peptide benzodiazepine-based ligands appear to occupy this putative pocket . Minor structural modifications either of these compounds or of the amino acids which comprise the receptor pocket , can influence whether these ligands act as agonists or antagonists. In addition to these findings with non-peptides, we have obtained evidence that affinity for the endogenous peptide agonist, gastrin, is conferred by an interaction between trans-membrane and extracellular domain amino acids. In the current application, we propose to further explore the molecular determinants of ligand binding to the CCK-BR, and of ligand-induced receptor activation, comparing peptide and non-peptide compounds. The relative roles of both the extracellular and transmembrane domains will be explored. Specific Aim 1 of this grant is directed toward defining CCK-BR amino acids which confer ligand affinity. Specific Aim 2 addresses which CCK-BR -ligand interactions influence the ability of the ligand to induce second messenger signaling. Specific Aim 3 will exploit the recent discoveries in our laboratory of a constitutively active CCK-BR together with a compound that functions as an inverse agonist and as such attenuates ligand-independent signaling. These novel tools will be utilized to explore the receptor-ligand interactions which result in inverse agonism. A combination of molecular (generation of chimeric and mutant receptors, transient expression of recombinant proteins) and pharmacologic methods (radioligand binding, second messenger signaling assays) will be utilized to address these objectives. The proposed studies of the CCK-BR will establish a framework for understanding how non-peptide ligands mimic the activity of endogenous peptides. This information should expand current knowledge of structure-function relationships of peptide hormone receptors in general and may therefore be useful in developing new therapeutic options for a wide range of diseases mediated by this class of proteins.